Phosphoinositides, Ezrin/Moesin, and rac1 Regulate Fusion of Rhodopsin Transport Carriers in Retinal Photoreceptors

Abstract

The post-Golgi trafficking of rhodopsin in photoreceptor cells is mediated by rhodopsin-bearing transport carriers (RTCs) and regulated by the small GTPase rab8. In this work, we took a combined pharmacological-proteomic approach to uncover new regulators of RTC trafficking toward the specialized light-sensitive organelle, the rod outer segment (ROS). We perturbed phospholipid synthesis by activating phospholipase D with sphingosine 1-phosphate (S1P) or inhibiting phosphatidic acid phosphohydrolase by propranolol (Ppl). S1P stimulated the overall rate of membrane trafficking toward the ROS. Ppl stimulated budding of RTCs, but blocked membrane delivery to the ROS. Ppl caused accumulation of RTCs in the vicinity of the fusion sites, suggesting a defect in tethering, similar to the previously described phenotype of the rab8T22N mutant. Proteomic analysis of RTCs accumulated upon Ppl treatment showed a significant decrease in phosphatidylinositol-4,5-bisphosphate-binding proteins ezrin and/or moesin. Ppl induced redistribution of moesin, actin and the small GTPase rac1 from RTCs into the cytosol. By confocal microscopy, ezrin/moesin and rac1 colocalized with rab8 on RTCs at the sites of their fusion with the plasma membrane; however, this distribution was lost upon Ppl treatment. Our data suggest that in photoreceptors phosphatidylinositol-4,5-bisphosphate, moesin, actin, and rac1 act in concert with rab8 to regulate tethering and fusion of RTCs. Consequentially, they are necessary for rhodopsin-laden membrane delivery to the ROS, thus controlling the critical steps in the biogenesis of the light-detecting organelle.

Rhodopsin C terminus, the site of mutations causing retinal disease, regulates trafficking by binding to ADP-ribosylation factor 4 (ARF4)

“…Alternatively, more than one ARF may crosslink to the rhodopsin C-terminal peptide. ARF3, which is present in the photoreceptor Golgi (31,34), is a potential candidate for this interaction.…”

“…The 20-kDa protein specifically crosslinked to peptide C and bound to SA-Sepharose was excised from a silver-stained gel and subjected to sequence analysis. Protein identification was performed at the Harvard Microchemistry Facility (Boston) by microcapillary reversephase HPLC nanoelectrospray tandem MS, as described (31). In addition to a tryptic fragment of frog rhodopsin C-terminal peptide (26), sequence analyses revealed one peptide unique to Xenopus ARF4 (A. L. Boman, National Center for Biotechnology Information, direct submission), accession no.…”

“…Confocal microscopy was performed on dark-adapted frog retinas, as described (31). Sections were labeled with monoclonal anti-rhodopsin mAb 11D5 (1:100), followed by Cy3 (red) goat anti-mouse IgG (1:200; Jackson ImmunoResearch), or with affinity purified anti-ARF4 (1:100), followed by Cy2 (green) goat anti-mouse IgG (1:200).…”

CD44 functions in Wnt signaling by regulating LRP6 localization and activation

“…On the one hand, ERMs regulate membrane delivery of proteins. 50 On the other hand, ERMs function as scaffold proteins that build molecular networks facilitating activation of proteins. 51 Our results show that CD44 is a positive regulator of Wnt signaling.…”

Merlin regulates transmembrane receptor accumulation and signaling at the plasma membrane in primary mouse Schwann cells and in human schwannomas

“…Interestingly, in a schwannoma cell line, merlin regulates membrane levels of PDGFR-b by inducing its degradation (Fraenzer et al, 2003), revealing an additional degree of complexity in merlin regulation of growth factor steady state levels. ERM proteins also are implicated in vesicular trafficking (Pujuguet et al, 2003;Deretic et al, 2004;Stanasila et al, 2006). Thus, merlin and ERM proteins regulate protein transport to and from the plasma membrane.…”